Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phospholipase C-mediated hydrolysis of phosphatidylcholine has been shown recently to be activated by a number of agonists. Muscarinic receptors, which trigger various signal transduction mechanisms including inhibition of adenylate cyclase through Gi, have been shown to be potent stimulants of this novel phospholipid degradative pathway. We demonstrate here, by exogenous addition of Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C, that phosphatidylcholine breakdown mimics the ability of carbachol to inhibit adenylate cyclase. This effect is sensitive to pertussis toxin and is entirely dependent on the presence of protein kinase C. This kinase is also required for the inhibition by carbachol of adenylate cyclase. These results suggest that the activation of phosphatidylcholine breakdown by phospholipase C may play an important role linking or favoring the coupling muscarinic receptors to Gi. Results presented here also show that phospholipase C-mediated hydrolysis of phosphoinositides by exogenous addition of Bacillus thuringiensis phosphoinositide-hydrolyzing phospholipase C does not affect adenylate cyclase, despite the fact that protein kinase C is translocated to an extent similar to that produced by the hydrolysis of phosphatidylcholine. According to the results shown here, both phospholipases also differ in their ability to down-regulate protein kinase C as well as to phosphorylate p80 and to transmodulate the binding of epidermal growth factor, two well established effects of protein kinase C in Swiss 3T3 fibroblasts. This emphasizes the complexity, from a functional point of view, of protein kinase C activation "in vivo."
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PMID:Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine. Involvement of a pertussis toxin-sensitive G protein and protein kinase C. 184 88

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.
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PMID:Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes. 184 16

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; also called phorbol myristate acetate) and epidermal growth factor both rapidly inhibit the transition of HeLa cells from G2 phase to mitosis in a reversible fashion with characteristics of a receptor-mediated response. It was proposed that an intracellular mediator was responsible for this inhibition. In searching for a common mediator elicited by the action of both ligands, the time course of generation of phospholipid metabolites was compared with the cell cycle response of the G2 cohort monitored by time lapse analysis. The time course and the degree of mobilization of diacylglycerols (DG) effected by TPA and by epidermal growth factor and that of phosphatidic acid correlated to a great extent with the onset and the duration of the G2 inhibition caused by both agonists. Further evidence for the proposed role of DGs and/or phosphatidic acid was obtained by the observation that membrane-penetrating 1,2-DG inhibited HeLa cells in G2 phase directly. The concentration of DG required correlated with that elicited by TPA or epidermal growth factor metabolically. This cellular response to 1,2-DG was also seen in cells after down-regulation of protein kinase C using TPA. The data indicate that cellular DG and/or phosphatidic acid may contribute to a restriction of cells in G2 phase possibly by changing membrane properties.
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PMID:A proposed common mechanism by which phorbol esters and epidermal growth factor delay the progression from G2 phase to mitosis of HeLa cells through phospholipid metabolites. 186 56

The increase in the intracellular concentration of Ca2+ in A431 cells induced by the calcium ionophore A23187 leads to phosphorylation of epidermal growth factor (EGF) receptors at serine and/or threonine residues. This process is accompanied by the decrease in the level of EGF receptor autophosphorylation at tyrosine residues. Preincubation of cells in a A23187-containing medium in the presence of phorbol-12-myristoyl-13-acetate leads to a further decrease of the phosphotyrosine content in EGF receptors. At increased intracellular concentrations of Ca2+ preincubation of A431 cells with the protein kinase C inhibitor H-7 has no effect on the degree of EGF receptor autophosphorylation. Down-regulation of cellular protein kinase C does not change the A23187-induced effect either. The data obtained suggest that the decreased autophosphorylation of EGF receptors induced by Ca2+ is not due to the activation of cellular protein kinase C.
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PMID:[Autophosphorylation of EGF receptors in the A431 cell line with an increased intracellular concentration of Ca(2+) ions]. 187 50

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.
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PMID:The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. 187 34

The epidermal growth factor (EGF) receptor is phosphorylated by protein kinase C at Thr654. It has been proposed that the phosphorylation of this site is an important regulatory mechanism for the control of EGF receptor function. However, the physiological significance of the phosphorylation of EGF receptor Thr654 in intact cells is not understood. To address this question, the design of an experimental strategy is required that can be used to distinguish between the pleiotropic effects of kinase C activation and the specific effects of kinase C that are mediated by the phosphorylation of the EGF receptor at Thr654. The approach that we used was to examine the function of EGF receptors that are constitutively phosphorylated at residue 654. It was observed that the constitutive phosphorylation of the EGF receptor blocked mitogenic signal transduction by the receptor. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at residue 654 in intact cells inhibits EGF-stimulated cellular proliferation.
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PMID:Constitutive phosphorylation of the epidermal growth factor receptor blocks mitogenic signal transduction. 189 31

Rapid stimulation of c-fos transcription by many agonists requires the serum response element (SRE), which binds at least two distinct nuclear proteins, p67SRF and p62TCF. Using nuclear protein extracts from 1321-N1 human astrocytoma cells, we investigated ligand-induced changes in binding of these proteins to SRE probes. In these cells c-fos mRNA expression can be induced by epidermal growth factor (EGF) through protein kinase C-independent pathways and by phorbol esters through protein kinase C. We detected two DNA-protein complexes that formed specifically with the SRE (bands 1 and 2). Band 2 formation was increased 4-6 min after stimulation with EGF as well as serum and phorbol esters; this peaked at 10-30 min and returned to basal levels by 60 min. Induction of band 2 formation preceded the onset and peak accumulation of c-fos mRNA (15 and 30 min after EGF stimulation, respectively) and its return to basal levels (by 1-2 h). Band 2 formation was also increased A431 cells stimulated with EGF and in HeLa and Swiss-3T3 cells stimulated with serum. We found that band 1 contained p67SRF bound to the SRE; band 2 contained p67SRF and a second protein. Gel shift analyses using [35S]methionine-labeled p67SRF and nonradioactive DNA probes suggested that hormone treatment most likely modified the second protein component of band 2. Transient transfection of 1321-N1 cells with plasmids containing point mutations that prevented band 2 formation in vitro also abolished induction of c-fos transcription in vivo as assayed by RNase protection analysis. Thus, hormone-stimulated formation of the protein-DNA complex represented by band 2 may be involved in the activation of c-fos transcription.
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PMID:Epidermal growth factor and other mitogens induce binding of a protein complex to the c-fos serum response element in human astrocytoma and other cells. 190 75

Employing the non-recirculating perfused rat liver preparation, we have investigated the regulation of hepatic gluconeogenesis, and metabolic fluxes through the tricarboxylic acid cycle and 2-oxoglutarate dehydrogenase reaction by epidermal growth factor (EGF) which mimics the actions of both insulin and Ca(2+)-mobilizing hormones (e.g. vasopressin). As monitored by the rate of 14CO2 production from [2-14C]pyruvate (0.5 mM), EGF (10 nM) transiently stimulated the activity of the tricarboxylic acid cycle. EGF also transiently stimulated hepatic gluconeogenesis from pyruvate. The transient stimulation of tricarboxylic acid cycle activity and gluconeogenesis were accompanied by an increase in perfusate Ca2+ content indicating that EGF also altered hepatic Ca2+ fluxes. EGF-elicited stimulation of gluconeogenesis was, at least in part, the result of a transient (50%) inhibition of pyruvate kinase activity. Likewise, EGF-mediated stimulation of tricarboxylic acid cycle activity can, in part, be attributed to EGF-elicited stimulation of metabolic flux through the mitochondrial, Ca(2+)-sensitive, 2-oxoglutarate dehydrogenase reaction. The regulation of hepatic metabolism by EGF appears to be the manifestation of alteration in cellular Ca2+ content since in experiments performed under conditions known to abolish the ability of EGF to alter cytosolic free-Ca2+ concentrations, i.e. in livers of pertussis-toxin-treated rats, EGF did not alter either perfusate Ca2+ content or any of the metabolic parameters monitored. Additionally, experiments involving pulsatile infusion of either EGF or phenylephrine into livers demonstrated that, unlike the alpha 1-adrenergic receptor, homologous desensitization of the EGF receptor occurs. Such a homologous desensitization of the EGF receptor can explain the transient nature of EGF-elicited stimulation of various metabolic processes. Since protein kinase C activation by EGF can lead to receptor desensitization, experiments were performed with phorbol esters which either activate or do not alter protein kinase C activity. While the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not modulate the hepatic actions of EGF, activation of protein kinase C by 4 beta-phorbol 12-myristate 13-acetate (70 nM) abolished the ability of EGF to stimulate gluconeogenesis, tricarboxylic acid cycle activity and metabolic flux through the 2-oxoglutarate dehydrogenase complex.
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PMID:Regulation of hepatic energy metabolism by epidermal growth factor. 190 8

The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate [TPA) or phorbol 12-myristate 13-acetate), directly activates the calcium- and phospholipid-dependent protein kinase C (protein kinase C), which, in turn, generates a number of cellular responses. The bryostatins, a family of macrocyclic lactones isolated from marine bryozoans, also bind to and active protein kinase C. However, they differ from TPA in the selectivity of their responses in that they behave either as agonists or antagonists of protein kinase C actions. We used several bryostatins and TPA to examine the role of protein kinase C in the regulation of GH4C1 rat pituitary tumor cell proliferation. TPA inhibited [3H]thymidine incorporation in GH4 cells in a stereoselective and concentration-dependent manner. Examination of cell cycle distribution by flow cytometry revealed that TPA decreased the percentage of cells in S-phase and proportionally increased the percentage of G1-phase cells. Bryostatin 1 alone did not affect cell proliferation, but prevented the TPA inhibition of cell proliferation. Bryostatin 1 treatment from 30 min to 6 h after TPA treatment also prevented the growth-inhibitory action of TPA, suggesting that prolonged stimulation of protein kinase C is necessary for growth inhibition. Both bryostatin 1 and TPA down-regulated protein kinase C, indicating that down regulation of the enzyme cannot account for the growth inhibitory action of TPA. Bryostatin 2, which differs from bryostatin 1 by a hydroxyl substitution for the acetyl group at the C-7 carbon of the macrocyclic lactone ring (R1), inhibited cell proliferation and did not reduce the growth-inhibitory action of TPA. Bryostatins 3 and 8 (each of which has an ester group in the R1 position, yet contains other structural modifications) are antagonists for TPA inhibition of GH4 cell proliferation like bryostatin 1. We next examined the effect of bryostatins 3 and 8 on cell-substratum adhesion, a cellular response observed after GH4 cells are treated with growth-inhibitory agents. Bryostatin 8 (like bryostatin 1) did not enhance cell-substratum adhesion and blocked the action of TPA. In contrast, bryostatin 3 enhanced cell-substratum adhesion. Because bryostatin 3 blocked TPA inhibition of cell proliferation, yet did not block TPA-enhanced cell-substratum adhesion, these responses are not interdependent. We next examined the effect of bryostatin on other growth-inhibitory agents for GH4 cells. Bryostatin 8 blocks the effect of TPA on [3H]thymidine incorporation and the entry of G1 cells into S-phase, but does not block the growth-inhibitory action of thyrotropin-releasing hormone or epidermal growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Bryostatins selectively regulate protein kinase C-mediated effects on GH4 cell proliferation. 190 62

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.
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PMID:Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation. 190 4


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